Dengue virus exploits the host tRNA epitranscriptome to promote viral replication.

The 40-50 RNA modifications of the epitranscriptome regulate posttranscriptional gene expression. Here we show that flaviviruses hijack the host tRNA epitranscriptome to promote expression of pro-viral proteins, with tRNA-modifying ALKBH1 acting as a host restriction factor in dengue virus infection. Early in the infection of human Huh-7 cells, ALKBH1 and its tRNA products 5-formylcytidine (f 5 C) and 2'- O -methyl-5-formylcytidine (f 5 Cm) were reduced. ALKBH1 knockdown mimicked viral infection, but caused increased viral NS3 protein levels during infection, while ALKBH1 overexpression reduced NS3 levels and viral replication, and increased f 5 C and f 5 Cm. Viral NS5, but not host FTSJ1, increased f 5 Cm levels late in infection. Consistent with reports of impaired decoding of leucine UUA codon by f 5 Cm-modified tRNA Leu(CAA) , ALKBH1 knockdown induced translation of UUA-deficient transcripts, most having pro-viral functions. Our findings support a dynamic ALKBH1/f 5 Cm axis during dengue infection, with virally-induced remodeling of the proteome by tRNA reprogramming and codon-biased translation.